Prostaglandins are derived from the oxygenation of arachidonic acid by prostaglandin (PG) synthases. Prostaglandins mediate a wide variety of physiological actions, such as vasomotricity, sleep/wake cycle, intestinal secretion, lipolysis, glomerular filtration, mast cell degranulation, neurotransmission, platelet aggregation, leuteolysis, myometrial contraction and labor, inflammation and arthritis, patent ductus arteriosus, cell growth and differentiation. Prostanoids mediate their actions through binding to distinct receptors which belong to the super family of rhodopsin-like seven transmembrane helical receptors. These receptors are coupled to heterotrimeric G-proteins comprised of α, β and γ subunits which, upon activation, elicit alterations in cell calcium, initiate phosphoinositide hydrolysis, or promotion or repression of cyclic adenosine monophosphate synthesis (Narumiya, S. et al. 1999; Physiol. Rev. 79: 1193-1226.).
Of the five pharmacologically-distinct prostanoid receptors PGE2, PGI2, PGD2, PGF2α, and TxA2, four subtypes of PGE2 receptor are described (Ichikawa, et al. 1996). These are EP1, EP2, EP3, which have several splice variants, and EP4. Cloned human EP4 (also known as prostaglandin E2 receptor subtype EP4) is a 488 amino acid glycoprotein, linked to GαS, and is involved in the stimulation of adenylate cyclase and cAMP synthesis (U.S. Pat. Nos. 5,759,789 and 5,605,814). The EP4 receptor is expressed at high levels in the intestine, but at much lower levels in the lung, kidney, thymus, uterus and brain (Bastien, Y. et al. 1994; J. Biol. Chem. 269 (16): 11873-77). The EP4 receptor is involved in fluid filtration in the kidney, differentiation of monocyte/macrophage precursors into osteoclasts, proliferation of intestinal crypt cells, and patency of ductus arteriosus in the mammalian fetus.
PGE2 is abundantly produced in the kidneys and is involved in the regulation of renal microcirculation, salt and water transport, and renin release (Breyer, M. D. et al. 1998; Kidney Int. 54 (Suppl. 67): S88-94). All EP receptors are regionally distributed in the kidney structures (Morath, R. et al. 1999; J. Am. Soc. Nephrol. 10: 1851-60) and are associated with specific functions. All studies conducted on the distribution of EP receptors in the kidneys have shown that the EP4 receptor is uniquely expressed in glomeruli (Breyer, M. D. et al. 1996; Am. J. Physiol. 270: F912-918. Morath, R. et al. 1999; J. Am. Soc. Nephrol. 10: 1851-60). However, the presence of this receptor in other structures of the nephron, such as the collecting duct (Breyer, M. D. et al. 1998; Kidney Int. 54 (Suppl. 67): S88-94), the media of renal arteries and vasa recta (Morath, R. et al. 1999; J. Am. Soc. Nephrol. 10: 1851-60) has been separately reported. EP4 transcripts have also been found in juxtaglomerular granule cells, and is consistent with PGE2-induced cAMP synthesis in these cells. EP4 may therefore also play a role in renin secretion.
Glomerular prostaglandins are thought to affect filtration (Schlondoff, D. et al., 1987; Kidney Int. 29: 108-19) and renin release. PGE2 increases cAMP levels in isolated glomeruli (Freidlander, G. et al., 1983; Mol. Cell. Endocrinol. 30: 201-214). It was suggested that the EP4 receptor coupled to cAMP synthesis, may regulate glomerular filtration (Sugimoto, Y. et al. 1994; Am. J. Physiol. 266(5 Pt 2):F823-8). Using small molecule antagonists (Kohno, Y. et al. WO 00/16760) and peptide antagonists (Peri, K. G. et al. WO 00/01445), a direct role of the EP4 receptor in modulating kidney filtration and urine output has been demonstrated.
Bones undergo continuous remodeling, wherein bone formation is carried out by osteoblasts and bone resorption is carried out by osteoclasts. These processes are controlled by several humoral factors such as parathyroid hormone, estradiol, vitamin D, cytokines, growth factors and prostaglandins. It has been illustrated that osteoclast induction by interleukin-1 (IL-1) is inhibited by aspirin-like drugs (Tai, H. et al. 1997). PGE2 analogues with EP4 receptor agonistic activity (no specific agonists or antagonists to this receptor exist to date) promote osteoclast formation in co-cultures of mouse osteoblasts and bone marrow cells. Similar experiments using cells from EP4-knockout mice resulted in reduced osteoclast formation, suggesting a role of the EP4 receptor in osteoclastogenesis in mice (Narumiya et al. 1999).
The ductus arteriosus is a normal large, low resistance, shunt vessel in fetuses, facilitating the bypass of blood towards the lungs. Since the fetus does not use its lungs (oxygen is provided through the mother's placenta), fetal lungs are collapsed and pose a high resistance to blood flow. Hence, blood flows from the right ventricle through the ductus into the descending aorta. High levels of circulating prostaglandins, particularly PGE2, keep the ductus in the foots open. When the infant is born, the lungs are inflated, the pulmonary resistance drops, PGE2 levels decrease, the ductus begins to close, and blood from the pulmonary artery thus enters into the lungs. The high levels of oxygen in the new born often close the ductus, in most cases within 24 hours. Patent Ductus Arteriosus (PDA) is the condition wherein the ductus doesn't close. In cases of PDA, morbidity and mortality rates are directly related to the flow volume through the ductus arteriosus. A large PDA may cause pulmonary hypertension, edema, recurrent infections, and may lead to congestive heart failure, if left untreated over long periods. Development of pulmonary vascular obstructive disease may occur. It is estimated that if left untreated, the mortality rate is 20% by the age of 20, 42% by the age of 45, and 60% by the age of 60. Females are 2 to 3 times more likely than males to develop PDA.
PDA can be treated either by drugs such as Indomethacin, which is a prostaglandin synthesis blocker, or by corrective surgery. Indomethacin, however, has side effects on renal ischemia and renal hypofusion, resulting in ischemic renal failure in preterm infants. EP4 is expressed in fetal pig (Bhattacharya, M. et al. 1999; Circulation 100(16): 1751-6), fetal lamb (Bouayad, A. et al., 2001; Am. J. Physiol. Heart Cir.c Physiol. 280(5); H2342-9) and fetal baboon (Smith G. C. et al., 2001; J. Cardiovasc. Pharmacol. 37(6): 697-704) ductus arteriosus. Paradoxically, EP4 knock-out mice die after birth due to insufficient closure of ductus arteriosus (Nguyen, M. et al. 1997; Nature, 390: 78-81).
A selective peptidic antagonist of the EP4 receptor has been used in the treatment of fetal ductus arteriosus (Peri, K. G. et al., WO 00/01445 and Wright, D. H. et al. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2001; 281 (5): R1343-60).
Prostaglandins, particularly PGE2, play an important role in intestinal crypt cell proliferation. In fact, the inducible prostaglandin synthesizing enzyme COX-2 was shown to be present in intestinal polyps, as well as in colon tumors (Shattuck-Brandt, R. L. et al., 1999; Mol. Carcinog. 24(3): 177-87). COX-2 selective blockers such as Nimesulide were used to prevent chemical induction of colon carcinogenesis (Jacoby, R. F. et al. 2000; Cancer Res. 60(18): 5040-4). Recently, the actions of PGE2 have been shown to be mediated by the EP4 receptor, as deduced from the low incidence of colon polyps in EP4−/− mice, due to azoxymethane and the efficacy of the EP4 selective antagonist ONO-AE2-227 in reducing aberrant crypt foci in azoxymethane-treated min mice (Mutoh, M. et al. 2002; Cancer Res. 62(1): 28-32).
There thus remains a need to develop selective peptide antagonists of the prostaglandin E2 receptor subtype EP4 and peptidomimetics thereof. More specifically, there remains a need to develop selective peptide antagonists of the prostaglandin E2 receptor subtype EP4 useful in the treatment and prevention of colon carcinogenesis, treating end-stage renal disease, acute renal failure and other conditions of renal insufficiency preventing bone resorption in osteoporosis, in addition to conditions preventing closing of the ductus (PDA) in the neonates.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.